The elemental composition of the 2001 attack anthrax presents critical clues that were not considered or were misinterpreted throughout the original investigation. Extensive experimental data released by the FBI after the anthrax case was closed make it possible to trace some of the implications of these clues: the substantial presence of tin, a toxic material that must have been added subsequent to growth, and a uniquely high content of silicon in the attack spores. No Bacillus spore preparations other than the attack anthrax have ever been found to contain such a high level of silicon, although some surrogate spore powders prepared at Dugway following FBI instructions have been cited as evidence that high levels of silicon can be reproduced; however, examination of the experimental data reveals that the silicon in these samples was unquestionably an artifact. The elemental evidence suggests that the attack spores had been coated with silicone (a polysiloxane) in the presence of tin, which catalyzes the cross-linking of polysiloxane chains needed to form an encapsulating coating on the spore coat. Microencapsulation helps protect biological agents from damage during atmospheric exposure and from the body’s defenses during infection, and would defeat some detection methods. Microencapsulation, which would explain the location and amounts of both tin and silicon in the attack spores, requires special expertise and sophisticated facilities. DOD-sponsored projects explicitly involving microencapsulation at DARPA, Dugway and perhaps elsewhere were spelled out publicly in budget documents in 1999 and thereafter, and executed at the very time of the anthrax attacks. Both the Dugway laboratory and Battelle Memorial Institute, a sub-contractor at Dugway, had extensive experience in making Bacillus spore powders; both had access to Bacillus anthracis genetically matching the attack spores; both could have made the attack spores legally for institutions conducting biodefense activities that required microencapsulated spores. Furthermore, a small but significant amount of tin, about 4% of that in the attack spores, has been found in some surrogate spore products made at Dugway. A measureable tin content has not been found in any other Bacillus spores except the attack spores. The tin in the Dugway surrogates may have been a remnant, indicative of earlier, classified work. Avoidance of governmentsponsored, classified research may account for some of the limitations of the investigation.

Introduction

The FBI’s investigation of the 2001 anthrax attacks focused heavily
on genetic evidence that strongly links the attack anthrax to a liquid
suspension of Ames-strain Bacillus anthracis spores in a flask known
as RMR 1029, found at USAMRIID (the US Army Medical Research
Institute for Infectious Diseases). Prior to the attacks, spores from this
flask − believed to be the parental source of the anthrax in the letters
– had been sent to a number of other laboratories. Collection and
analysis of an FBI repository of samples from all laboratories known to
possess the Ames strain indicated that several laboratories had Bacillus anthracis that was identical or nearly identical to that in RMR 1029.
There is no public information, however, as to whether any of those
laboratories may have produced the attack anthrax, possibly as part of
their authorized work1.

In addition to the genetic composition of the attack anthrax, the
elemental compositions of the spore powders provide equally important
clues: the presence of tin, a toxic material that must have been added
subsequent to growth; and a uniquely high content of silicon, not in the
familiar form of silica nanoparticles. Examination of the extensive (but
incomplete) experimental data released by the FBI in February 2011
and of other evidence from government sources has made it possible to
trace some of the implications of these clues2. It is remarkable that there
is no evidence that the FBI tried to determine the chemical forms of
the tin and silicon in the attack powders, although that could have been
(and still could be) done straightforwardly by spectroscopic methods.
Perhaps the FBI knows the answers but has concealed them in order to
keep potentially dangerous information out of the hands of adversaries.

Significance of tin in the attack anthrax

Analysis by the FBI for tin in the attack samples, using ICP-OES
(inductively-coupled plasma optical emission spectrometry), found
0.1979 wt% tin in the Leahy powder and 0.6511 wt% in the NY Post
sample3. In contrast, many other Bacillus spore preparations4 from
various sources that were subjected to elemental analysis by the FBI or
its contractors were found to contain no tin at all (see data in Table 1,
and summary of tin contents in Table 2).

To explain the substantial presence of tin in the attack spores, which
has never been addressed by the FBI, Hugh-Jones et al.5 have proposed
that the spores were processed after growth by coating them with silicone,
typically a polysiloxane formed by hydrolysis and polymerization of a
silane compound, in the presence of tin. Tin catalyzes the cross-linking
of polysiloxane chains, which would thereby form an encapsulating
silicone coating on the spore coats6 – the location at which tin, and
silicon as well, have been found7 in the attack spores. This process, which would explain the presence, location and amounts of both tin
and silicon, would require special expertise and sophisticated facilities.
It is an aim of this paper to explore the evidence that the spores used
in the letter attacks may have been microencapsulated for legitimate
biodefensive purposes before they fell into the hands of the letter
sender(s).

“Reverse engineering” of the silicon content of the attack
spores

Natural incorporation of silicon by Bacilli, from silicates in growth
media, was first reported in 19808. Sandia National Laboratory has
shown that the silicon in samples made at that time, as well as Bacillus
samples more recently produced in other laboratories, is located at
the spore coat9. All of the many Bacillus anthracis and other Bacillus
spore preparations with naturally-incorporated silicon that have been
analyzed by the FBI or its contractors have contained a maximum
of 0.5 wt% silicon, usually much less, often zero (Table 1), with the
sole exception of eight samples from a set of 36 agar-grown Bacillus anthracis spore powders prepared in 2003 at Dugway (DPG, Dugway
Proving Ground) according to FBI instructions, in an effort to
“reverse engineer” the letter powders (data in Table 1, silicon contents
summarized in Table 3). The 36 samples comprised all permutations of:
two media, two washing procedures, four drying procedures, and three
milling procedures10. Based on the plan and the extensive experimental
data provided by Dugway11, there appears to have been no intentional
effort to reproduce the silicon or tin in the attack spores; the emphasis
seems to have been on particle size and viability. However, at that time
there was considerable public concern about the silicon in the attack
spores (the presence of tin had not been divulged by the FBI).

The FBI selected only ten of these “surrogate” samples for elemental
analysis, using ICP-OES in an FBI laboratory. Eight of the ten samples
were found to contain unusually high levels of silicon (0.5-5 wt%, average 1.4 wt%)12. These eight are the only known Bacillus preparations, other
than the attack spores, containing more than 0.5 wt% silicon. These
eight samples were all ball-milled, while the remaining two samples,
which contained only 0.2 and 0.3 wt% silicon, were milled by hand in a
porcelain mortar and pestle13,14. The FBI did not choose to examine any
of the samples that had been milled by a third procedure – a manual
stainless steel ball and sieve method.

The ball-milled samples also contained extremely high levels of
aluminum (0.4-3 wt%), while the mortar-and-pestle-milled samples
contained only 0.0087 and 0.0149 wt% aluminum15 (The NY Post
and Leahy samples contained 0.0158 and 0.0220 wt% aluminum,
respectively).

The ball mill described by Dugway consisted of a porcelain milling
jar containing a grinding medium consisting of zirconium cylinders,
rotated for approximately 16 hours. Porcelain is composed of silica and
alumina. The presence of a high level of aluminum only in the ballmilled
samples is telltale and would be hard to miss. Furthermore, the
Dugway laboratory noted that all ball-milled samples (but not those
milled by other methods) showed a high degree of clumping, as viewed
microscopically, making spore viability determination impossible16, and
the FBI laboratory noted that the Dugway samples were abnormal in not
fully responding to the usual acid digestion procedure, as a consequence
of which the concentrations of two of the samples in particular-those
with the highest silicon content (5 and 2 wt%) – were likely inflated17.
These observations signal that most or all of the silicon and aluminum
in the eight ball-milled samples was an extra-sporular artifact. Analyses
done at Sandia of two of the ball-milled Dugway preparations (those
containing 5 and 0.7% wt% silicon, NDLB and SDLB in Table 1), using
STEM-EDX (scanning electron microscopy with energy-dispersive
X-ray analysis) measurements on thin sections, confirm that none of
the silicon was in the spore coat18, where it is located both in the attack
spores and in Bacilli containing naturally-incorporated silicon19.

The question arises as to why the FBI wanted any of the Dugway
samples to be ball-milled, or milled at all, given that the purpose of
making the samples was “to give some insight into how the materials in
the [anthrax] letters were made20”, and given that the Dugway samples
were made in 2003, well after Battelle (Battelle Memorial Institute, BMI,
a defense contractor) had prepared unmilled Bacillus globigii surrogates
that compared favorably to the Leahy attack spores with regard to
dispersibility, as indicated by aerosol particle size distribution (see next
section). Battelle’s report to the FBI on particle sizes, dated February
2002, noted that their result “indicates that neither milling nor other
processing of a freeze-dried B. anthracis spore powder was required21”.

The FBI/Dugway sample preparation plan22 had called for some
additional samples to be dried by an (undescribed) acetone method in
use at Dugway, but this part of the plan was evidently set aside, although
portions of pastes from two other samples were eventually acetonedried23
– but they were never named, further processed, described or
analyzed, at least not in publicly-released documents. Acetone drying
might have obviated any reason for milling: Bruce Ivins, who assisted
the FBI in the early part of the investigation, later quoted one of his
USAMRIID colleagues as telling him “he’d use an organic solvent like
acetone or alcohol to pull water out of purified spores and then easily
make them into powder24”.

Along with the 36 agar-grown samples, Dugway had also
prepared two surrogate samples by fermentation in liquid Leighton-
Doi medium25. This was an interesting experiment, in which one
fermentation sample was grown in the presence of an antifoam agent
containing polydimethylsiloxane (Sigma Antifoam C), while the other
was grown with an antifoam containing no silicon (Sigma Antifoam
204). Did the spores that had been exposed to polydimethylsiloxane
during growth contain a higher level of silicon than has been found
in spores exposed only to silicates in media? If so, might the attack
spores have been exposed to polydimethylsiloxane? The only relevant information released to the NAS Committee and the public was Sandia’s
observation that a Dugway fermentation sample received from the FBI
under the code name “040255-1,” for determination of silicon in the
spore coats, contained some silicon on about 25% of the spore coats26.
This information was used solely as evidence that some fermentation
samples contain some silicon. Which of the two fermentation samples
this was, and how much silicon was on the spore coats and in the bulk
sample, were not revealed to the NAS or the public. Sandia noted,
however, that, although some of the various surrogate samples they
studied contained silicon on the spore coat, compared to the attack
samples “the details are different…chemistry, distribution27”.

The silicon content of the eight ball-milled Dugway agar-grown
surrogates has been wrongly cited as evidence that high levels of silicon
can be reproduced in Bacillus anthracis preparations28. Similarly, the fact
that small amounts of silicon can sometimes be incorporated naturally
into the coats of Bacillus spores during their growth has been cited
to imply that the much larger amounts of silicon in the attack spore
powders must result from the same process29, even though no growth
experiments have actually reproduced the high silicon content, nor has
the chemical nature of the silicon in the two cases been determined and
compared. This selective use of partial information may have convinced
many who have not looked into the experimental details that the silicon
question has been resolved.

Because of the suspicion that the silicon in the attack spores
might have been there to increase their aerosol dispersibility, Battelle
– known for its aerosol expertise – was asked to compare the aerosol
particle size distributions of the attack spores to those of dry simulant
spore preparations made at Battelle without any special post-growth
processing30. The simulants were two Bacillus globigii powders, “washed”
and “unwashed,” isolated only by centrifugation, water-washing (or
not) and lyophilization31. The aerosol particle size distributions of
the Bacillus globigii samples were found to be bimodal, with 1-2% in
the single spore range; their distributions were similar to, and slightly
narrower than, that of a sample taken directly from the Leahy letter and
analyzed in February 200232. The similarity of the results for the Leahy
and surrogate samples appears to confirm the FBI’s repeated insistence
that the attack spores had not been treated in any way that affected
aerosol dispersibility. These results can best be understood in light of
the observations of Dr. Thomas Geisbert at USAMRIID, who measured
particle sizes in the pristine Daschle sample by electron microscopy
and reported that hydrated samples consisted of single spores – which
accounts for the Daschle titer of 2.1×1012 cfu/g, the theoretical limit for
pure, 100% viable, single Ames spores; but in dry samples (such as those
used for aerosol studies) Geisbert found that the spores aggregated to
form clumps of up to 105 spores33.

Battelle initially (October 2001) examined the aerosol particle
size distributions of two Daschle powders provided by the FBI right
after the attack, labeled SPS.57.0334 and SPS.57.0835, neither of which
was taken directly from the attack letter itself36. These were the results
that were most frequently cited by the FBI. Both the Daschle samples
had very low titers (4.6×1010 cfu/g and 1.5×108 cfu/g, respectively) and
showed other signs of contamination and aggregation37. It is therefore
not surprising that the particle size results for these samples compare
poorly with the Bacillus globigii results. Why did the FBI choose to send
samples recovered from spills in Daschle’s office, rather than a pristine
sample, for this important early test? Only the Leahy data, obtained some
four months later and largely ignored, can be considered significant,
allowing the tentative conclusion that the attack spores probably had no
special advantage or disadvantage for aerosol dispersion.

Microencapsulation of pathogens in US biodefense research

Although there is no evidence to indicate that the tin and silicon
content of the spores conferred any benefit for purposes of the letter
attacks, their presence is meaningful if the attack spores had been
prepared legitimately for other purposes. Silicone microencapsulation would have been desirable for increasing the resistance of the spores
to inactivation by hazards such as UV light, ozone or toxic materials38,
and for preventing detection of the spores by some methods. (It is at
the spore coat, rather than the external membrane (the exosporium)
of Bacillus anthracis that these functions occur39). These are properties
of military concern. The use of microencapsulation for such purposes
was already an old idea40 in the biodefense community in the years
immediately preceding the attacks. Microencapsulation by special
polymers to produce particles in the 1-10 micron range could protect
microbes from environmental damage during aerosolization and
delivery [e.g. via bomblets] and also from the body’s initial defenses
during the infection process, according to an encyclopedia on weapons
of mass destruction published in 2004, and could also help defeat
some detection schemes41. The encyclopedia notes that the technology
requires an advanced research and development infrastructure,
unlikely to be available to terrorists, but “state-level CBW programs
could certainly employ microencapsulation to produce highly effective
weapons of mass destruction42”.

In the non-military literature it has recently been shown that
single, living Bacillus spores can be encapsulated using layer-by-layer
polyelectrolyte nanocoating43, and that individual cells, including
Bacillus spores, can then be subsequently encapsulated with silica44;
the silica encapsulation greatly enhanced viability by protecting the cell
from harsh environments.

By 1999 and thereafter, DOD (Department of Defense) interest
in pathogen encapsulation was explicitly spelled out in (unclassified)
budget documents for the Biological Warfare Defense program at
DARPA (the Defense Advanced Research Projects Agency) and the
Chemical/Biological Defense program, which includes Dugway.

DARPA’s Biological Warfare Defense program was conducting a
project, initiated in 1995, to develop a miniature time-of-flight mass
spectrometer for rapid detection of a broad spectrum of chemical and
biological warfare agents in aerosol form45. Johns Hopkins University’s Applied Physics Laboratory (APL) took the lead, with critical
collaboration from USAMRIID to provide and test pathogens and
develop a database of their mass spectral signatures; this work included
preparation of both Sterne46 and Ames (inactivated) Bacillus anthracis
powders – the latter grown from an RMR 1012 inoculum in late 200047.
Dr. John Ezzell of USAMRIID, who acted as the FBI’s scientific advisor
on anthrax, speaking from the floor at a seminar on the Amerithrax
investigation on Nov. 29, 2010, described his production of (sterile)
anthrax spores at that time, commenting that the powders produced in
his lab were purer than any of the letter powders48 and that high purity
was needed for determining unique mass spectral signatures49,50.

From 1999 to 2001, annual DOD budget item justifications for
the DARPA project on detectors (called “sensors”) listed a plan to
be carried out in FY 2001 to “evaluate methods for removing microencapsulation
of disguised pathogens and/or sensing through the
micro-encapsulation51”. The budget item justification sheet dated
February 200252 listed that plan under “FY2001 Accomplishments”,
indicating that at least one microencapsulated pathogen was studied
by DARPA in 2001.

Also accomplished by DARPA in 2001 was the evaluation of
“methods of cell stabilization for possible application to cell based
sensors”. One such stabilization method is microencapsulation.

Concomitantly, the Chemical/Biological Defense Program
(CBDP)53, covering work at Dugway, undertook in 1999 to identify and
evaluate emerging threat agents by various means, and continued work
on this project through FY2001, during which year they assessed the
gaps in the threat agent data and the needs for improved simulants. Also
in 2001, plans were made to initiate a program of synthesis, toxicology screening and characterization of new threat materials, to include
Fourth Generation Agents [which include those altered for better
survival54, e.g. by microencapsulation], and to initiate development
of improved simulants for microencapsulated viruses and stabilized
bacteria. Throughout this period the program provided controlled
biosimulant aerosol challenges for Joint Service, DARPA, and DOE
experimental equipment. Dugway tested DARPA’s detection equipment
in 199955.

Because of Dugway’s emphasis on improved simulants and on
stabilization/survivability, Dugway may be the source of the unique
B. subtilis contaminant found in the early attack letters56. There is no
evidence that Dugway or any other site was ever examined for the
presence of the critical Bacillussubtilisstrain. The strain was found to
be a hypersporulator57, typical of the strains that originated at Dugway
for use as simulants58. The Bacillussubtilis isolated from the NY Post
letter was sequenced for the FBI59 and found to have greater than 98%
identity60 with the widely-studied strain Bacillussubtilis 168 (sequenced
in 199761), often used as a surrogate for Bacillus anthracis62 and as the
standard model in studies of the molecular and genetic basis of Bacillus
spore resistance to environmental stresses63. Research on Bacillus
genetics was proliferating at the time of the anthrax attacks.

Dugway is the only place known to have made live, dry, weaponsgrade
anthrax powder in the years before the attacks64. During the
Amerithrax investigation the Dugway laboratory was the place the
FBI asked to conduct experiments attempting to reverse-engineer
production of the attack anthrax powders. The Dugway laboratory had
supplied USAMRIID with most of the spores in flask RMR 1029, the
putative parental source of the attack anthrax, in 1997.

A B. anthracis stock sample provided by Dugway to the FBI Ames
anthrax repository tested positive in at least one of the four genetic
assays used as indicators of relationship to the attack anthrax65, and the
NAS committee believed that Dugway probably produced all four of the
genetic markers used for assays66. A subcontractor67 working at Dugway
in 2001, Battelle Memorial Institute, had twice received material from
RMR 1029 from USAMRIID during 200168, upon receipt of which
Battelle was given permission “to provide aliquots to other laboratory
facilities for legitimate research purposes69”.

Battelle is well-known for aerosol expertise, and was working
with dry anthrax simulant spores in the period before the attacks70. It
would probably be difficult to distinguish whether Dugway or Battelle
personnel were responsible for any anthrax-related work done at
Dugway at that time.

A new clue

FBI documents released in 2011 show that the ten “reverse
engineered” surrogate samples made at Dugway for the FBI contained
a small but significant amount of tin. The FBI has never commented
on this finding, nor on the tin content of the attack spores, and there
is no evidence that the “reverse engineering” experiments71 or any
other investigations were aimed at reproducing or explaining the
presence of tin. No other Bacillus spore preparations except the attack
spores contained tin (Table 2). The tin content of the Dugway samples,
measured by ICP-OES in the FBI laboratory72, varied from 0.0033-
0.0266 wt% (average 0.0086 wt% tin). Omitting the two samples for
which the FBI laboratory reported that the analytical results were most
questionable73 gives a range of 0.0033-0.0084 (average 0.0058 wt%).
In any case, the Dugway surrogates contained about 3-4% as much
tin as was found in the Leahy spores (0.197 wt%) (data in Table 1). A
“commercially available multi-element standard solution74” analyzed at the same time had 0.0069 wt% (69 ppm) tin75. The presence of tin
cannot be attributed to ball milling; the two samples that were not ballmilled
contained 0.0038 and 0.0064 wt% tin, consistent with the tin in
the eight ball-milled samples. No tin was found in any culture medium
components76 nor was tin exposure likely during growth or in any of the
post-growth treatments77.

Random, trace contaminants are extremely unlikely to be found
by ICP-OES in the concentration range observed in the Dugway
samples. Analyses of a set of Bacillus thuringiensis samples at Sandia
National Laboratory using TOF-SIMS (time of flight secondary ion
mass spectrometry), a more sensitive method than ICP-OES78, found
traces of tin, too small to quantify, in those samples79. One of the Sandia
authors (J. Michael) is quoted80 as saying “we were surprised at first [at
the traces of foreign elements], then we realized that the elements could
have come from any number of sources − lab equipment, a residual
cleaning solution, some other kind of contamination”. Contaminants
that were not quantifiable by TOF-SIMS would certainly not be
observable or quantifiable by ICP-OES81.

The small but real tin content found by the FBI laboratory in the
Dugway samples appears to have been overlooked. Was it a remnant of
tin used previously at Dugway in classified work? There is no evidence
whatsoever to rule out the possibility that the attack samples had been
legally made at Dugway82 or elsewhere. It must be recognized, of course,
that making the spores is not synonymous with sending the letters. The
“attack” anthrax could have been made for the use of US agencies or
contractors conducting legitimate activities such as vulnerability and
response assessment or testing detection devices such as DARPA’s. The
letter sender(s) may have been one or more individuals who, whether
legally or illegally, had access to the material at some point in the
process.

Discussion

Tin, found by the FBI in substantial amounts on the spore coats
of the attack anthrax, has not been discussed or investigated. Tin is toxic to bacteria and therefore must have been acquired by the attack
spores after their growth. The quantities are too high to be accidental
contaminants. In approximately the amounts found83, tin is known to be
a catalyst in the formation of silicone coatings for microencapsulation.
Although there may be other possible explanations for the presence
of tin, neither the FBI nor anyone else has put forward an alternative.
The microencapsulation hypothesis is strengthened by the evidence
that government-sponsored programs specifically involving
microencapsulated pathogens were in progress at the very time of the
attacks.

Silicon, in uniquely high amount, was also found on the coats of the
attack spores. Although the FBI reported similarly high silicon content
in a set of surrogate Bacillus anthracis spore powder preparations
“reverse engineered” at Dugway, examination of the laboratory data
(released after the case was closed) reveals that the silicon present was
unquestionably a milling artifact and had no connection to the spores.
Neither the Dugway surrogate spores nor any of the many other Bacillus
spore preparations examined have ever been found to contain levels of
silicon comparable to the attack spores (Table 3). These facts cast strong
doubt on the FBI’s explanation that the silicon in the attack spores was
naturally incorporated during growth. The FBI’s investigation of the
silicon issue has been characterized by loose assumptions and sketchy
data, raising fundamental questions about the investigation’s approach
to the whole matter of additives in the attack spores.

The presence, shown by FBI analysis, of the two extraneous
elements, tin and silicon, together in the attack spores favors the
silicone microencapsulation hypothesis. Microencapsulation, a process
that would require special expertise and sophisticated facilities, could
explain the presence, location and amounts of both elements. At least
two government programs, at DARPA and Dugway, had projects
requiring microencapsulated pathogens or simulants. Both Dugway
and Battelle, a sub-contractor there, had access to Bacillus anthracis
from the presumptive parental flask RMR 1029. Both had the expertise
to make anthrax spore powders, both – and perhaps other government- supported laboratories as well – could have made the attack spores
legally for institutions conducting biodefense activities that required
microencapsulated spores.

Other than the attack spores, no Bacillus preparations have ever
been found to contain tin, with one exception: the set of ten Bacillus anthracis powders “reverse engineered” at Dugway in 2003 (Tables 1
and 3). These samples were produced by standard, well-described
preparation procedures, with no known exposure to tin. Nonetheless,
FBI analyses that became available after the case closed found a small
but real amount of tin in the Dugway powders, about 4% of the
amount in the attack spores, too large to be attributed to random, trace
contamination; contamination from a previous use of tin appears to
be the most likely explanation. The tin in the Dugway samples may be
an indicator of previous, classified work with tin at Dugway to provide
materials for biodefense activities.

Many US agencies were involved in biological antiterrorism activities
at the time of the anthrax attacks. The Chemical and Biological Defense
Program initiated in FY 2000 a “broad CB countermeasures program to
enhance ability to recognize, prevent, respond to, mitigate, and recover
from a CB terrorist incident84”. There had already been an epidemic
of hoax anthrax letters. CIA scientists possessed Ames anthrax and
were working with other government agencies and outside contractors
[including Battelle85] in the defensive biowarfare program86; former
biological weaponeer Bill Patrick wrote a classified paper about anthrax
sent by mail, dated February 1999, for SAIC (Science Applications
International Corporation) under CIA contract87. Nonetheless, the CIA
issued a statement that they were unaware of any project to assess the
impact of anthrax sent through the mail88; similarly, the FBI claimed to
have been blindsided by a letter attack89.

The evidence indicates that live, microencapsulated Bacillus anthracis spore powders probably existed legitimately at the time of the
attacks and were the likely sources of the anthrax in the attack letters.
A number of individuals would have had access to such materials,
whether legally or illegally. For the letter-sender(s), the presence of
additives in the powders would have been irrelevant. For the FBI, a real
investigation of the presence of additives may have been impossible
without ‘off-limits’ intrusion into classified biodefense matters90.

2Sets of FBI Documents identified by B (Batch) and M (Module) numbers were provided by the FBI to the National Academy of Sciences (NAS) Committee on the Review
of the Scientific Approaches Used During the FBI’s Investigation of the 2001 Bacillus anthracis Mailings Investigation; the documents are listed in the NAS Report issued
February 15, 2011, pp. 133ff, Index of Documents Provided by the Federal Bureau of Investigation; the documents were released to the public in February 2011 and were
available from the NAS at that time.

4The only exception, a set of 10 “reverse engineered” samples made at Dugway, is discussed in a later section.

5Hugh-Jones et al., op. cit.

6See Hugh-Jones et al. (op. cit.) for details of the proposed siliconization procedure, which requires moisture from within the spores and results in deposition on the spore
coat, not on the exterior surface of the spore (the exosporium).

13The milling methods used by Dugway for the 36 samples are described in FBI Document B1M13, p. 71.

14The mortar-and-pestle- milled samples, labeled NDLM (0.3 wt% silicon) and NPLM (0.2 wt%), can be compared to their ball-milled counterparts differing only in milling
method: NDLB (5 wt% silicon) and NPLB (0.5 wt%). Note that NDLB was one of the two samples for which the analytical results were questionable, according to the FBI
laboratory report (B1M7, p. 28); the other was SPVB (2 wt%). These two are the samples with the highest analytical results for silicon.

15FBI Document B1M7, FBI Elemental Analysis Summary, p. 93.

16FBI Document B1M13 (Dugway), pp. 23 and 84-85, where it can be seen that all 12 of the ball-milled Dugway samples, not just those analyzed by the FBI, exhibited
clumping.

17FBI Document B1M7 (FBI Laboratories), p. 28.

18NAS Report “Review of the Scientific Approaches used during the FBI’s Investigation of the 2001 Anthrax Letters,” Feb. 15, 2011, p. 67 (analysis) and p. 70 (identity of
the samples studied).

19Michael J, Kotula P (2009), op. cit.; also Sandia reports to the FBI in FBI documents B1M6 and B1M1 (in these reports, samples are coded and it is sometimes difficult
or impossible to find their identities).

20FBI Document B1M13 (Dugway), p. 93.

21FBI Document B2M13 (Battelle), p. 151.

22FBI Document B1M13 (Dugway), Test Plan, p. 15.

23FBI Document B1M13 (Dugway), p. 79.

24Amerithrax Investigative Summary, February 19, 2010, p. 75.

25B1M13 (Dugway), pp. 70 and 74-5. The two fermentation products, grown in 2003 at the same time and from the same stock as the agar samples, were stored until 2005,
when they were irradiated and processed (methods not disclosed) under the designations “Lot 05AUG05” for one grown with Antifoam 204 and “Lot 01SEP05” for one
grown with Antifoam C.

26FBI document B1M1(Technical Review Panels), Sandia report on elemental mapping of Amerithrax Samples, pp. 109, 110, 100, 106. The data for this sample, coded
040255-1, in Sandia’s Table on page 109 are the same as those for the “Dugway surrogate (fermentation using Leighton-Doi media)” in the NAS Report “Review of the
Scientific Approaches Used During the FBI’s Investigation of the 2001 Anthrax Letters,” Feb. 15, 2011, pp 67 and 70, indicating that the FBI must have identified the coded
material to the NAS Committee as a Dugway fermentation sample, but evidently did not specify which one.

31FBI Document B2M13 (Chemical and Physical Characteristics, Battelle), The Analysis of Surrogate Dry Powder Bacillus Spore Product, pp. 92ff. The “unwashed” preparation
had a titer of 9×1011 cfu/g, an indication of its high quality (compare to the titer of the pristine Daschle sample, 2.1×1012, which had been determined at USAMRIID just
after the material was received from the Capitol Police (FBI Document B1M2 (USAMRIID), Anaytical Test Report, pp. 36ff; note pp. 37 and 42). The titer of spores in the
Leahy letter has never been reported.

32FBI Document B2M13 (Chemical and Physical Characteristics, Battelle), Summary of Sample Analyses, pp. 146ff. Analysis of the Leahy sample was done in February
2002, after the Leahy letter had been stored in a mail bag for a number of months; it is therefore possible that the Leahy spores were no longer in their original condition.

47On August 28, 2000, forty ml were removed from flask RMR 1029 at USAMRIID “for DARPA mass spec project with JHU-APL” (inventory control sheet, www.vault.fbi.gov/
amerithrax, part 24, p. 8); see also, FBI interview believed to be of Dr. Joany Jackman, a major participant in the DARPA project working at USAMRIID under John Ezzell
from 1997-2000 and then at Johns Hopkins APL: B. anthracis, grown from an inoculum (about 1012 cfu/ml) provided by Bruce Ivins and purified on a gradient, was used
at USAMRIID for the aerosol work (http://vault.fbi.gov/Amerithrax/ part 21, pp 19-22; Jackman’s corroborating biographical details are at http://www.hopkinsmedicine.org/
medicine/std/team/Jackman.html).

64New York Times, US Recently Produced Anthrax in a Highly Lethal Powder Form, December 13, 2001.

65FBI Document B2M10 (Statistical Analysis), especially Appendix V of the Report on Statistical Analysis, where the names of some specific laboratories that submitted
repository samples of interest are handwritten next to the sample data: DPG (Dugway Proving Ground); BMI (Battelle Memorial Institute); DRES (Defense Research Establishment,
Suffield, Canada); NMRC (Naval Medical Research Center); USAMRIID. See also the National Academy of Sciences (NAS) Report “Review of the Scientific
Approaches used during the FBI’s Investigation of the 2001 Anthrax Letters,” February 15, 2011, pp. 110-112, and FBI Document B2M10, p. 25. From information in these
documents it appears that seven repository samples from USAMRIID and one from Battelle tested positive in all four genetic assays; one sample each from USAMRIID and
Northern Arizona University (an FBI contractor in the anthrax case) had three positives; a samples from DRES had two positives; and samples from six other laboratories,
including Dugway and the Naval Medical Research Center plus four unnamed laboratories, tested positive in one or two assays, as did additional samples from some of
the laboratories already mentioned.

66NAS Report (op. cit.), p. 108 gives reasons why all four markers probably originated at Dugway; the Report also discusses the probability of false negatives and presents
a Table of assay results on 30 repeat samplings of flask RMR 1029 (the putative parental source of the attack anthrax) as an illustration (p. 117).

67Personal communication January 11, 2002 from David Lore, Columbus Dispatch Science Reporter, author of article “Labs deny use of letter anthrax: Powdered spores
not part of stocks, Battelle official says” in Columbus Dispatch, January 9, 2002.

68FBI Document B3D16. One Ames repository sample from Battelle tested positive in all 4 assays; another from Battelle, known to have originated from an RMR 1029
sample, tested positive in 2 of 3 assays (FBI Document B2M10, p. 25).

69Maureen Stevens et al. vs United States of America: Notice of Errata, Document 162, entered on FLSD Docket 07/19/2011, submitted by the Defendant United States in
US District Court, Southern District of Florida, Case Number: 03-81110-CIV-Hurley/Hopkins.

70Columbus Dispatch, Labs deny use of letter anthrax: Powdered spores not part of stocks, Battelle official says, January 9, 2002.

71See FBI/Dugway plan for the reverse engineering work in FBI Document B1M13.

72Elemental analyses of the 10 samples, measured by ICP-OES in an FBI laboratory, are given in FBI Document B1M7 (FBI Laboratory Reports), Samples from Dugway,
pp. 26-28; the data are repeated at B1M7, Elemental Analysis Summary, p. 93.

75FBI Document B1M1 (Technical Review Panels), p. 83, gives the elemental analysis of the Standard, together with averages for the elements in the ten Dugway samples
(presented at a Chemistry Review Panel in August 2005).

82The finding, via stable isotope analysis (B1M9, p.44), that Dugway water is unlikely to have been used to grow the attack spores is probably not relevant; Dugway’s report
(B1M13) on preparation of the surrogate samples mentions the use of “sterile water for irrigation” and “sterile water for injection,” which are generally purchased in small
bottles from distant providers. The FBI laboratory, in analysing media components (B1M7), included “Baxter sterile water for irrigation”.

90An appropriately targetted investigation would seek to determine whether Bacillus anthracis had been microencapsulated prior to the letter attacks, and, if so, by whom,
where, when, and the amounts, strains and dispositions of the resulting materials. Parts of such an investigation might still need to be classified.